November  l>7,  190y. 


o==-       U.  S.  DEPARTMENT  OF  AGRICULTURE, 

*  BUREAU  OF  ANIMAL  INDUSTRY— BULLETIN  121. 


A.  D.  MELVIN,  CHIEF  OF  BUREAU. 


7         IE    NEED  OF  CONTROLLING  AND   STANDARDIZING 
THE  MANUFACTURE  OF  VETERINARY 
TETANUS  ANTITOXIN. 


BY 
JOHN  R.  MOHLER,  V.  M.  D., 

Chief  of  the  Pathological  Diinsion, 
AND 

ADOLPH  EICHHORN,  D.  V.  S., 

Bacteriologist  in  the  Pathological  Division. 


WASHINGTON: 

GOVERNMENT    PRINTING   OFFICE. 

1909. 


Issued  November  27,  1909. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY— BULLETIN  <\2<\. 
A.  D.  MELVIN,  CHIEF  op  BUREAU. 


THE    NEED  OF  CONTROLLING  AND   STANDARDIZING 

THE  MANUFACTURE  OF  VETERINARY 

TETANUS  ANTITOXIN. 


BY 

JOHN  R.  MOHLER,  V.  M.  D., 

Chief  of  the  Pathological  Division, 
AND 

ADOLPH  EICHHORN,  D.  V.  S., 

Bacteriologist  in  the  Pathological  Division. 


WASHINGTON: 

GOVERNMENT    PRINTING   OFFICE. 

1909. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  MELVIN. 
Assistant  Chief:  A.M.  FARRINGTON. 
Chief  Clerk:  CHARLES  C.  CARROLL. 
Biochemic  Division:  M.  DORSET,  chief. 
Dairy  Division:  B.  H.  RAWL,  chief. 

Inspection  Division:  RICE  P.  STEDDOM,  chief;  MORRIS  WOODEN,  R.  A.  RAMSAY, 
and  ALBERT  E.  BEHNKE,  associate  chiefs. 
Pathological  Division:  JOHN  R.  MOHLER,  chief. 
Quarantine  Division:  RICHARD  W.  HICKMAN,  chief. 
Zoological  Division:  B.  H.  RANSOM,  chief. 
Experiment  Station:  E.  C.  SCHROEDER,  superintendent. 
Animal  Husbandman:  GEORGE  M.  ROMMEL. 
Editor:  JAMES  M.  PICKENS. 


LETTER  OF  TRANSMITTAL. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY, 

Washington,  D.  C.,  November  2, 1909. 

SIR:  I  have  the  honor  to  transmit  herewith  and  to  recommend  for 
publication  in  the  bulletin  series  of  this  Bureau  a  manuscript  entitled 
"The  Need  of  Controlling  and  Standardizing  the  Manufacture  of 
Veterinary  Tetanus  Antitoxin,"  by  Dr.  John  R.  Mohler,  chief  of  the 
Pathological  Division  of  this  Bureau,  and  Dr.  Adolph  Eichhorn, 
bacteriologist  in  that  division. 

The  investigation  reported  in  this  paper  was  undertaken  in  accord- 
ance with  the  act  of  Congress  making  appropriations  for  the  Depart- 
ment of  Agriculture  for  the  past  fiscal  year,  which  empowered  the 
Secretary  of  Agriculture  to  purchase  in  the  open  market  samples  of 
tuberculin,  serums,  antitoxins,  etc.,  designed  for  veterinary  use,  to 
test  the  same,  and  to  publish  the  results  of  such  tests.  This  act  does 
not,  however,  confer  any  authority  to  supervise  or  control  the  manu- 
facture of  these  products. 

It  has  become  known  in  the  veterinary  profession  that  tetanus 
antitoxin  for  veterinary  use — unlike  that  for  human  use,  which  is 
manufactured  under  stringent  government  supervision — is  urgently 
in  need  of  standardization.  Under  existing  conditions  the  veter- 
inarian and  the  stock  owner  are  at  the  mercy  of  the  manufacturers 
of  this  antitoxin,  some  of  whom  make  no  statement  as  to  the  strength 
of  their  product,  while  in  the  great  majority  of  the  cases  tested  in  the 
present  investigation  it  was  found  that  the  standard  was  not  reached 
and  many  of  the  samples  were  far  below  it. 

It  is  very  necessary  that  the  veterinarian  should  have  some  reliable 
assurance  of  the  strength  of  this  most  valuable  therapeutic  agent. 
There  is,  therefore,  pressing  need  for  legislation  empowering  the 
Secretary  of  Agriculture  to  supervise  and  control  the  manufacture 
of  veterinary  tetanus  antitoxins  and  to  prescribe  and  enforce  a 
proper  standard  of  potency.  Such  authority,  of  course,  would  be 
limited  to  products  manufactured  for  interstate  commerce  or  offered 
for  importation. 

Very  respectfully,  A.  D.  MELVIX, 

Chief  of  Bureau. 

Hon.  JAMES  WILSON, 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Page. 

Introduction 7 

Nature  and  cause  of  tetanus 8 

Historical  summary 9 

Mode  of  action  of  tetanus  toxin 11 

Toxicity  of  tetanus  toxin 12 

Stability  of  tetanus  toxin 12 

The  standardization  of  tetanus  antitoxin 13 

European  methods 13 

The  American  method  of  standardization 14 

Examination  of  commercial  veterinary  antitoxins 15 

Conclusions 22 

5 


THE  NEED  OF   CONTROLLING   AND   STANDARDIZING 

THE  MANUFACTURE  OF  VETERINARY 

TETANUS  ANTITOXIN. 


INTRODUCTION. 

Of  the  various  biological  products  prepared  for  the  cure  and  pre- 
vention of  infective  diseases  in  animals  tetanus  antitoxin  has  been 
probably  the  most  extensively  used  by  the  veterinary  profession. 
The  good  effects  which  were  expected  from  the  administration  of 
this  antitoxin  in  the  prophylactic  and  curative  treatment  of  tetanus 
have  not,  however,  been  all  that  could  be  desired,  and  it  is  surprising 
to  learn  of  the  divergent  results  which  are  obtained  from  its  use  by 
various  practitioners.  It  is  therefore  only  natural  that  tetanus  anti- 
toxin has  not  gained  the  favor  of  the  veterinarian  and  is  not  used  to 
the  extent  merited  by  such  a  valuable  product.  It  has  been  sug- 
gested that  the  lack  of  uniform  results  which  has  followed  the  admin- 
istration of  the  antitoxin  is  primarily  due  to  the  variation  in  the 
strength  of  the  product,  and  following  numerous  requests  from  dif- 
ferent sources  it  was  decided  to  undertake  the  standardization  of 
veterinary  tetanus  antitoxins  prepared  by  different  manufacturers, 
and  also  to  determine  whether  these  products  are  subject  to  any 
variations  in  strength.  This  work  seemed  particularly  desirable  in 
view  of  the  address  of  Anderson  before  the  June  meeting  of  the 
American  Medical  Association,  in  which  he  stated  that  his  experi- 
ments showed  veterinary  tetanus  antitoxin  to  contain  as  low  as  17 
to  25  antitoxin  units  per  cubic  centimeter,  while  similar  examinations 
of  antitoxin  prepared  for  medical  use  contained  from  150  units  to 
600  or  even  700  units  per  cubic  centimeter. 

The  investigation  herein  recorded  was  carried  out  in  accordance 
with  an  act  of  Congress  making  appropriations  for  the  Department 
of  Agriculture  for  the  year  ending  June  30,  1909,  which  provides  as 
follows : 

That  the  Secretary  of  Agriculture  is  authorized  to  purchase  in  the  open  market 
samples  of  all  tuberculin,  serums,  antitoxins,  or  analogous  products,  of  foreign  or 
domestic  manufacture,  which  are  sold  in  the  United  States  for  the  detection,  pre- 
vention, treatment,  or  cure  of  diseases  of  domestic  animals,  to  test  the  same,  and  to 
publish  the  results  of  said  tests  in  such  a  manner  as  he  may  deem  best. 


8  VETERINARY   TETANUS  ANTITOXIN. 

It  should  be  noticed  that  while  power  has  been  granted  to  purchase, 
test,  and  publish  upon  these  products,  no  authority  has  been  given 
with  reference  to  the  supervision  and  control  of  their  manufacture. 
This  paper  is  presented  with  the  object  of  furnishing  some  concrete 
examples  of  the  variation  observed  in  tetanus  antitoxin  at  present  on 
the  market,  and  to  show  the  necessity  for  Federal  supervision  of  all 
vaccines,  serums,  antitoxins,  viruses,  and  analogous  products,  includ- 
ing mallein,  tuberculin,  anthrax  and  blackleg  vaccine,  and  hog-cholera 
serum. 

NATURE  AND  CAUSE  OF  TETANUS. 

Tetanus  is  an  extremely  painful  disease  of  all  animals,  including 
man,  but  it  more  particularly  affects  the  horse,  ass,  and  mule.  It  is 
caused  by  the  Bacillus  tetani.  This  organism  differs  from  other 
disease-producing  bacteria  in  the  fact  that  it  remains  localized  at 
the  point  of  entrance  to  the  body,  which  is  usually  through  a  wound. 
Here  it  multiplies  and  produces  a  powerful  toxin  which  is  absorbed 
into  the  circulation  fcud,  by  acting  on  the  nervous  system,  gives  rise 
to  the  symptoms  of  the  disease.  From  this  it  will  be  seen  that 
tetanus  is  the  type  of  the  purely  toxic  disease  and  is  not  transmissible 
by  contact  unless  the  wound  containing  the  germ  is  discharging  pus 
or  blood,  as  may  be  the  case  in  tetanus  following  the  operation  of 
castration.  Neither  can  the  disease  be  transmitted  from  one  animal 
to  another  by  blood  inoculation,  because  no  bacilli  are  circulating  in 
the  blood.  The  bacillus  is  a  spore  former,  and  these  spores  are  very 
resistant  to  antiseptics.  It  can  live  outside  the  animal  body,  and  is 
quite  prevalent  in  garden  earth  and  in  the  neighborhood  of  manure 
piles.  It  is  an  anaerobic  organism  like  the  bacillus  of  blackleg,  and 
consequently  can  multiply  only  in  the  absence  of  oxygen.  Deep 
punctured  wounds  about  the  feet  and  legs  are  especially  serious,  as 
in  wounds  of  this  kind  the  bacilli  are  most  likely  to  be  implanted, 
and  once  they  have  gained  access  the  absence  of  oxygen  in  these 
deep  wounds  makes  the  conditions  favorable  for  their  growth.  The 
period  of  incubation  varies  from  a  few  days  to  two  weeks. 

The  symptoms  come  on  gradually.  There  is  stiffness  of  the  gait 
and  rigidity  of  the  muscles,  the  appearance  of  the  animal  somewhat 
resembling  a  horse  foundered  in  all  four  feet.  The  neck  is  rigid,  the 
head  extended,  and  the  tail  elevated  and  immovable.  The  charac- 
teristic symptom  of  tetanus  is  the  protrusion  of  the  nictitans  mem- 
brane over  the  eye  when  the  head  is  raised.  This  condition  is  due 
to  a  contraction  of  the  muscles  of  the  eyeball  drawing  it  back  into 
its  socket  and  forcing  out  the  nictitans  membrane.  There  is  no  other 
disease  in  which  this  condition  is  present.  The  animal  is  extremely 
nervous,  starts  at  the  slightest  sound,  loses  its  appetite,  and  sweats 


HISTORY   OF   TETANUS   INVESTIGATIONS.  9 

freely.  There  is  a  spasmodic  contraction  of  certain  groups  of  mus- 
cles, especially  those  of  the  jaw,  so  that  the  animal  is  unable  to  open 
its  mouth,  from  which  the  disease  derives  its  popular  name  of  lock- 
jaw. The  malady  runs  an  irregular  course  and  death  may  occur  in  a 
few  days  or  the  affection  may  be  prolonged  over  two  or  three  weeks. 
Spontaneous  recovery  is  very  doubtful,  and  the  outlook  is  always 
grave.  The  mortality  for  some  classes  of  animals  has  been  given  as 
almost  100  per  cent,  while  about  75  per  cent  of  the  cases  in  horses 
terminate  fatally.  Unfortunately  the  disease  is  seldom  suspected 
until  a  relatively  large  amount  of  toxin  has  formed  and  begun  to 
manifest  its  action  in  the  patient's  body. 

HISTORICAL    SUMMARY. 

Prior  to  the  discovery  of  the  cause  of  tetanus  there  were  various 
theories  advanced  regarding  the  character  of  the  disease.  One  of 
the  most  widely  accepted  views  was  that  tetanus,  since  it  generally 
develops  as  a  result  of  an  injury,  was  occasioned  by  the  tearing  or 
contusion  of  some  of  the  peripheral  nerves  and  as  a  consequence  the 
changes  produced  were  conveyed  to  the  spinal  cord.  On  the  other 
hand,  in  the  cases  where  no  injury  was  associated  with  the  disease  it 
was  thought  to  be  of  idiopathic  origin. 

With  the  discovery  of  the  tetanus  bacillus  by  Nicolaier  in  1884 
and  its  successful  cultivation  by  Kitasato  in  1889,  the  true  cause  of 
the  disease,  which  previously  had  been  involved  in  mystery,  was 
established.  Kitasato  in  his  investigations  also  found  that  the  or- 
ganism is  not  present  in  the  blood  of  animals  dying  of  the  affection, 
and  accordingly  he  concluded  that  the  fatal  results  were  produced 
by  an  intoxication  and  not  by  the  infection.  In  his  subsequent  work 
with  this  disease  he,  together  with  Von  Behring,  successfully  worked 
out  and  published  an  immunizing  method  in  1890. 

It  has  already  been  stated  that  in  tetanus,  unlike  most  of  the  other 
infectious  diseases,  the  organism  itself  does  not  exert  the  destructive 
influence,  only  the  toxins  of  the  organisms  being  responsible  for  the 
serious  results  of  the  disease.  The  tetanus  bacilli  therefore  produce 
a  specific  substance  which  has  the  toxic  effect.  Kitasato  came  to 
this  conclusion  in  the  course  of  his  experiment  when  he  succeeded  in 
producing  typical  cases  of  tetanus  in  mice,  guinea  pigs,  rabbits,  and 
other  animals  with  the  filtrate  obtained  by  filtering  tetanus  cultures 
through  porcelain  filters.  At  the  same  time  he  found  that  it  required 
only  a  remarkably  small  quantity  of  the  toxin  to  produce  tetanus 
with  a  fatal  termination  in  these  animals 

In  the  above-mentioned  epoch-making  work  of  Von  Behring  and 
Kitasato  they  attributed  the  established  immunity  to  the  effect  of 

13477— Bull.  121—09— 


10  .  VETERINARY   TETANUS   ANTITOXIN. 

the  blood  serum,  which  rendered  harmless  the  toxic  substances  pro- 
duced by  the  tetanus  bacillus.  From  the  following  described  ex- 
periments they  obtained  results  demonstrating  that  the  serum  had 
an  enormous  power  of  destroying  the  poison: 

Of  a  tetanus  culture  10  days  old  which  was  freed  from  bacilli  by 
filtration,  0.00005  c.  c.  was  sufficient  to  kill  a  mouse  in  four  to  six 
days,  and  0.0001  c.  c.  was  sufficient  to  kill  a  mouse  with  certainty  in 
two  days.  However,  5  c.  c.  of  the  serum  from  the  tetanus-immune 
rabbits  were  mixed  with  1  c.  c.  of  this  culture  and  the  serum  was 
allowed  to  act  upon  it  for  twenty-four  hours.  Four  mice  were  each 
given  0.2  c.  c.  of  the  mixture,  which  contained  0.0033  c.  c.  of  the  cul- 
ture, or  more  than  300  times  the  dose  otherwise  fatal  for  mice.  All 
four  of  the  mice  remained  well.  The  control  mice,  on  the  other  hand, 
died  in  thirty-six  hours  after  having  received  0.0001  c.  c.  of  the 
fluid.  As  a  result  of  these  and  other  experiments  the  authors  drew 
the  following  conclusions: 

1.  The  blood  of  tetanus-immune  rabbits  possesses  the  property  of  destroying  the 
tetanus  poison. 

2.  This  property  is  destroyed  by  the  extra  vascular  blood  and  the  cell-free  serum 
obtained  from  it. 

3.  This  property  is  of  such  a  stable  nature  that  it  is  also  effective  in  the  bodies  of 
other  animals,  so  that  we  are  in  position  to  accomplish  noteworthy  therapeutic  results 
by  means  of  the  transfer  of  blood  serum. 

4.  The  property  of  destroying  the  tetanus  poison  is  absent  in  the  blood  of  animals 
which  have  not  been  immunized  against  tetanus.     If  tetanus  be  given  to  susceptible 
animals  the  toxin  may  be  demonstrated  in  the  blood  and  other  body  fluids  after  the 
death  of  the  animals. 

Tizzoni  and  Cattain  have  also  established  that  the  blood  of  artifi- 
cially immunized  pigeons  and  dogs  has  a  protective  action  on  mice  and 
rats  against  the  tetanus  toxin  (1  to  2  drops  of  the  dog  serum  made 
0.5  c.  c.  of  a  toxic  culture  filtrate  ineffective). 

With  these  wonderful  results  as  a  foundation,  the  serum  therapy 
of  tetanus  was  established. 

The  practical  application  of  this  method  was  inaugurated  after 
Schutz  had  found  that  horses  and  sheep  can  also  be  successfully 
immunized  against  tetanus  and  that  they  produce  an  active  immu- 
nizing serum.  Horses  are  now  exclusively  used  for  the  production 
of  tetanus  antitoxin,  as  large  quantities  of  blood  may  be  drawn  from 
an  immunized  horse,  which  constitutes  a  great  advantage  in  the 
manufacturing  of  this  product  for  the  market. 

The  immunizing  serum  is  prepared  according  to  Von  Behring's 
method  in  the  following  way:  Of  200  c.  c.  of  a  virulent  bouillon 
culture,  of  which  0.75  c.  c.  kills  a  rabbit  in  three  to  four  days,  80  c.  c. 
is  mixed  with  sufficient  trichlorid  of  iodin  to  make  a  0.25  per  cent 
solution;  similarly,  60  c.  c.  is  mixed  with  enough  to  make  a  0.175 
per  cent  solution,  and  40  c.  c.  with  sufficient  to  make  a  0.125  per 


ACTION    OF   TETANUS   TOXIN.  11 

cent  solution,  while  the  remaining  20  c.  c.  of  the  culture  is  left  without 
any  addition.  The  horse  is  then  injected  every  eight  days,  com- 
mencing with  10  to  20  c.  c.  of  the  greatest  dilution  and  continuing 
successively  with  the  weaker  attenuations  of  the  culture.  Finally 
the  pure  culture  is  injected,  commencing  with  0.5  c.  c.  and  doubling 
the  dose  every  five  days,  until  no  reaction  to  the  culture  is  manifested 
and  the  blood  shows  the  highest  number  of  immunity  units  obtainable 
from  that  individual  animal. 

MODE    OF    ACTION   OF   TETANUS    TOXIN. 

The  highly  poisonous  qualities  of  this  toxin  and  the  fact  that  the 
organism  causing  the  disease  acts  only  through  the  generated  toxin 
induced  a  greater  number  of  investigators  to  carry  out  various 
investigations  in  connection  therewith.  The  tetanus  bacillus,  it  will 
be  remembered,  does  not  enter  the  general  organism,  but  remains  at 
the  point  of  entrance  (wounds)  and  only  exerts  its  destructive  effect 
through  the  action  of  the  toxins  which  it  eliminates.  From  the  place 
of  infection  the  toxin  is  conveyed  principally  by  the  nerves  to  the 
central  nervous  system,  and  here  is  its  point  of  attack.  The  central 
nervous  system,  and  especially  the  spinal  cord,  is  first  affected  by 
the  toxin,  and  all  functional  manifestations  of  the  intoxication,  as 
well  as  the  increased  reflex  irritability,  such  as  the  tonic  spasms  of 
the  muscles,  are  the  result  of  the  poisoning  of  the  nerve  cells  of  the 
spinal  cord.  That  the  muscle  itself  is  not  directly  influenced  in  its 
function  by  the  tetanic  poison  is  no  longer  doubted  by  pathologists. 

The  tetanus  toxin  is  taken  up  by  the  peripheral  nerve  endings  and 
is  then  conducted  by  the  nerves  to  the  medullary  centers,  where  it 
combines  with  the  motor  nerve  cells.  The  poisoning  of  these  cells 
then  produces  the  tonic  muscular  spasms  as  well  as  the  reflex  irrita- 
bility (Meyer  and  Ransom,  Bruschettini,  Stinzing,  Von  Behring,  and 
others) . 

The  mode  of  action  of  the  tetanus  toxin  is  explained  by  Ehrlich's 
side-chain  theory  in  the  following  way:  Every  toxin  molecule  con- 
sists of  a  nonpoisonous  (haptophore)  and  a  poisonous  (toxaphore) 
atom  group.  On  the  other  hand,  the  protoplasm  of  the  motor 
nerve  cells  consists  of  a  vital  nucleus  and  of  numerous  side  chains 
(receptors),  of  which  some  possess  a  special  affinity  toward  the 
haptophore  atom  groups  of  the  toxin  molecules.  If  molecules  of  the 
tetanus  poison  enter  the  nerve  cells,  they  become  anchored  to  the 
corresponding  receptors  of  the  cell  protoplasm  by  the  haptophore 
atom  group,  and  their  toxophore  group  exerts  a  harmful  effect  on 
the  vital  nucleus  of  the  cell.  There  follows  either  a  total  destruction 
of  the  cell  protoplasm  as  a  result  of  this  combination  or  a  stimulation 
of  the  cell  protoplasm  to  defensive  action  which  is  manifested  by  the 
continual  production  and  elimination  of  the  receptors  into  the  blood. 


12  VETERINARY   TETANUS  ANTITOXIN. 

The  presence  of  these  free  receptors  in  the  blood  of  immunized  ani- 
mals explains  the  antitoxic  immunity,  since  these  receptors  are  enabled 
to  combine  with  the  haptophore  atom  of  the  toxin  molecule  before 
it  has  had  an  opportunity  to  attack  the  cell  protoplasm. 

The  tetanus  toxin,  according  to  Ehrlich,  contains  at  least  two 
poisons,  the  tetano-lysin  and  the  tetano-spasmin.  He  showed  that 
on  the  relative  proportions  of  these  poisons  depends  whether  the 
toxin  has  stronger  tetanic  properties  and  a  weaker  hemolytic  action, 
or  vice  versa. 

TOXICITY   OF   TETANUS    TOXIN. 

Testing  the  tetanus  toxin  for  its  virulence,  Kitasato  found  that 
0.0002  c.  c.  of  the  filtrate  of  a  tetanus  culture  proved  fatal  to  a 
mouse,  while  0.002  c.  c.  killed  a  guinea  pig  weighing  560  grams. 
A  rabbit,  on  the  other  hand,  required  relatively  double  the  quantity 
of  the  mouse  and  almost  seven  times  as  much  as  the  guinea  pig,  since 
it  required  0.04  c.  c.  of  the  filtrate  to  kill  a  rabbit  weighing  1,490 
grams.  Accordingly,  of  these  three  species  of  experimental  animals 
the  guinea  pigs  are  the  most  susceptible  to  tetanus  toxin,  mice 
coming  next  and  rabbits  last.  Of  all  animals  the  horse  is  probably 
the  most  susceptible  to  this  toxin. 

The  remarkable  poisoning  qualities  of  tetanus  toxin  can  be  seen 
from  the  findings  of  Briszer  and  Cohn;  0.00000005  gram  of  their 
strongest  tetanus  toxin  killed  mice  weighing  15  grams.  Smaller 
doses,  as  0.00000001  gram,  caused  tetanic  symptoms  in  mice.  The 
minimal  lethal  dose  (MLD)  of  the  standard  tetanus  toxin  of  the 
Hygienic  Laboratory  of  the  United  States  Public  Health  and  Marine- 
Hospital  Service  is  0.000006  gram  for  a  guinea  pig  weighing  350 
grams. 

STABILITY   OF   TETANUS    TOXIN. 

Kitasato  was  the  first  to  make  extensive  tests  on  the  stability  of 
the  tetanus  toxin,  and  while  other  investigators  have  since  under- 
taken similar  work  with  it  the  results  they  have  obtained  merely 
confirmed  the  findings  of  Kitasato. 

In  experimenting  upon  the  effect  of  physical  influences  on  the  fil- 
trate, Kitasato  found  that  tetanus  toxin  is  quite  susceptible  to  heat. 
Subjected  to  65°  C.  and  above,  it  is  totally  destroyed  in  five  minutes, 
while  it  resists  60°  for  fifteen  minutes,  but  twenty  minutes'  expos- 
ure to  this  temperature  destroys  it.  The  drying  of  the  filtrate  does 
not  necessarily  destroy  its  virulence.  However,  a  great  deal  depends 
upon  the  method  of  drying,  as  for  instance,  if  dried  in  the  incubator  it 
is  entirely  destroyed,  while  if  dried  in  the  exsiccator  over  sulphuric  acid 
or  in  the  air  at  room  temperature  it  retains  its  virulence.  Exposure 
of  the  filtrate  to  diffused  light  showed  a  gradual  diminishing  effect 


METHODS   OF   STANDARDIZATION.  13 

upon  its  toxic  action;  however,  it  requires  a  long  time  before  its 
virulence  is  entirely  destroyed,  as  the  nitrate  which  had  been  exposed 
for  nine  to  ten  weeks  to  the  diffused  light  in  the  window  still  had  toxic 
effects  in  large  doses.  On  the  other  hand,  the  filtrate  retains  its 
virulence  for  a  very  long  period  if  kept  cold  in  a  dark  room,  since  it 
did  not  lose  any  of  its  virulence  after  three  hundred  days  under  such 
conditions.  Exposure  of  the  filtrate  to  direct  sunlight  proved  that 
it  is  entirely  destroyed  only  after  fifteen  to  eighteen  hours,  while 
action  of  the  filtrate.  Dilutions  of  the  filtrate  with  water  or  bouillon 
shorter  exposure  showed  a  corresponding  diminution  of  the  toxic 
does  not  influence  the  virulence  of  the  tetanus  poison. 

Of  the  chemical  influences  on  the  filtrate  Kitasato  found  that 
tetanus  toxin  is  very  susceptible  to  the  action  of  acids  (especially 
mineral  acids)  as  well  as  alkalies. 

Practically  the  same  results  were  obtained  by  Rosenau  and  Ander- 
son regarding  the  stability  of  the  tetanus  toxin,  and  as  a  result  of 
their  extensive  investigation  with  the  dried  toxin  they  found  that 
it  not  only  retains  its  virulence  when  kept  in  a  cold,  dark  place,  but 
loses  its  toxicity  quite  slowly  when  exposed  to  light,  heat,  and  other 
influences.  One  sealed  tube  sent  from  Washington  to  Manila  by 
mail  arrived  there  without  appreciable  loss  of  toxicity.  The  sta- 
bility of  the  dried  toxin  is  of  great  advantage  in  the  work  of  stand- 
ardization of  the  antitoxins,  as  it  assures  accurate  work  and  also  sim- 
plifies and  expedites  the  tests. 

THE    STANDARDIZATION   OF   TETANUS    ANTITOXIN. 
EUROPEAN    METHODS. 

With  the  establishment  of  the  principles  of  immunizing  against 
tetanus  by  Von  Behring  and  Kitasato,  it  became  necessary  to  adopt 
a  method  by  which  the  potency  of  the  antitoxin  could  be  accurately 
determined.  In  Germany  the  testing  of  the  tetanus  antitoxin  is 
carried  out  in  accordance  with  the  Von  Behring  method,  which  pro- 
vides that  normal  serum  shall  be  a  serum  of  which  0.1  c.  c.  renders 
ineffective  0.03  gram  of  normal  toxin.  This  normal  or  test  toxin  is 
a  dried  toxin,  1  gram  of  which  has  a  virulence  capable  of  killing 
10,000,000  mice  each  weighing  15  grams.  The  Italian  method  of 
standardizing  tetanus  antitoxin  is  based  on  the  work  of  Tizzoni  and 
Cattain.  The  toxin  unit,  according  to  this  method,  is  the  smallest 
amount  of  toxin  which  will  kill  a  rabbit  weighing  1  kilogram  in  from 
four  to  five  days  The  Tizzoni  antitoxin  contains  80,000  immunity 
units  in  every  cubic  centimeter;  in  other  words,  this  amount  of  anti- 
toxin will  neutralize  80,000  toxin  units. 

The  French  method  of  standardizing  tetanus  antitoxin  is  carried 
out  by  the  subcutaneous  inoculation  of  a  series  of  guinea  pigs  with 


14  VETERINARY   TETANUS  ANTITOXIN. 


quantities  of  serum  equal  to  30V?nnr»  looioTro,  TeotUoo,  etc.,  of  their 
weight.  Twenty  hours  later  these  test  animals  are  given  a  single 
fatal  dose  of  toxin  and  the  immunizing  power  of  the  serum  is  then 
considered  to  be  T7  ATT  or  TTnrBTnnr>  etc.,  dependent  upon  whether 
the  animal  receiving  this  proportional  weight  of  serum  has  survived 
the  toxin  which  proved  fatal  to  the  control  guinea  pig. 

THE   AMERICAN    METHOD   OF   STANDARDIZATION. 

The  method  of  standardization  which  was  carried  out  in  connection 
with  the  testing  of  the  veterinary  antitoxins  as  reported  in  this  paper 
followed  the  exact  lines  of  the  method  known  as  the  American 
method,  which  has  been  adopted  officially  under  the  biological 
product  act  of  July  1,  1902. 

This  method,  which  is  the  result  of  several  years'  work  on  this  sub- 
ject in  the  Hygienic  Laboratory,  is  a  highly  creditable  achievement 
by  Rosenau  and  Anderson,*  as  it  not  only  simplifies  the  standardi- 
zation of  this  valuable  serum,  but  is  also  perfectly  reliable  in  its  accu- 
racy. The  method  was  also  unanimously  adopted  by  the  Society  of 
American  Bacteriologists  subsequent  to  the  following  report  made 
by  a  special  committee  : 

That  tetanus  antitoxin  be  standardized  by  the  tetanus  toxin  furnished  by  the  Public 
Health  and  Marine-Hospital  Service.  The  unit  is  ten  times  the  least  amount  of  serum 
necessary  to  save  the  life  of  a  350-gram  guinea  pig  for  ninety-six  hours  against  the 
official  test  dose  of  the  standard  toxin.  The  test  dose  is  100  minimal  lethal  doses  of  a 
precipitated  toxin  preserved  under  special  conditions  at  the  Hygienic  Laboratory  of 
the  Public  Health  and  Marine-Hospital  Service.  It  was  decided  that  the  minimal 
immunizing  dose  for  a  case  of  possible  infection  through  a  wound  should  be  1,500  of 
such  units.  It  was  decided  that  after  April  1  the  new  unit  should  be  adopted  by  all 
producers  of  tetanus  antitoxin. 

J.  J.  KINYOUN,  Chairman. 
HERBERT  D.  PEASE, 

JOSEPH   McFARLAND, 

THEOBALD  SMITH, 

E.  M.  HOUGHTON, 

M.  J.  ROSENAU, 

WILLIAM  H.  PARK,  Secretary. 

In  this  method  the  immunity  unit  for  measuring  the  strength  of 
tetanus  antitoxin  is  fixed  so  that  it  shall  be  ten  times  the  least  quan- 
tity of  antitoxin  serum  necessary  to  save  the  life  of  a  350-gram  guinea 
pig  for  ninety-six  hours  against  the  official  test  dose  of  a  standard 
toxin  furnished  by  the  Hygienic  Laboratory  of  the  Public  Health 
and  Marine-Hospital  Service. 

Thus  it  is  required  from  the  manufacturers  of  the  "human"  tetanus 
antitoxin  to  state  the  number  of  units  their  products  contain,  which 

a  Rosenau  and  Anderson.  The  standardization  of  tetanus  antitoxin.  Bulletin  43, 
Hygienic  Laboratory,  U.  S.  Public  Health  and  Marine-Hospital  Service.  Wash- 
ington, 1908. 


EXAMINATION   OF   COMMERCIAL  ANTITOXINS.  15 

not  only  insures  serum  of  reliable  strength,  but  also  establishes  a 
uniformity  among  the  producers  of  tetanus  antitoxin  in  America.  On 
the  other  hand,  the  antitoxins  destined  for  "veterinary"  use  are  still 
under  no  control  whatsoever;  there  is  no  uniformity  in  the  method  of 
standardization,  and  the  potency  of  the  product  is  absolutely  left  to 
the  honesty  of  the  manufacturer.  This  state  of  affairs  of  course  does 
not  assure  the  veterinarian  of  any  uniformity  of  the  product  even  of 
the  same  manufacturer,  still  less  of  different  manufacturers.  Only 
one  of  the  different  veterinary  tetanus  antitoxins  on  the  market  states 
the  number  of  American  units  the  immunizing  or  curative  dose  con- 
tains. The  others  fail  to  make  any  declaration,  and  in  one  case  the 
number  of  units  is  given  in  the  hundred  thousands,  whereas  the  human 
antitoxin  of  the  same  firm  of  course  complies  with  the  requirements 
of  the  law,  stating  definitely  the  number  of  American  units  that  par- 
ticular antitoxin  contains.  These  conditions  alone  should  suffice  to 
point  out  the  necessity  for  supervision  of  this  veterinary  product  and 
the  establishment  of  uniformity  among  the  producers  of  veterinary 
antitoxin.  It  is  very  essential  that  the  veterinarian  should  have  some 
assurance  of  the  strength  of  the  antitoxin  upon  which  his  standing  as 
a  professional  man  may  depend. 

EXAMINATION  OF  COMMERCIAL  VETERINARY    ANTITOXINS. 

The  samples  of  tetanus  antitoxin  (veterinary)  which  were  stand- 
ardized in  connection  with  this  work  were  obtained  from  various  drug 
stores  in  Chicago,  New  York,  and  Washington.  They  were  kept  at  a 
temperature  from  50°  to  60°  C.  until  the  tests  were  made,  care  being 
taken  not  to  expose  them  to  any  condition  which  might  affect  the 
potency  of  the  antitoxin. 

The  toxin  was  obtained  from  the  Hygienic  Laboratory  of  the  Public 
Health  and  Marine-Hospital  Service  through  the  courtesy  of  Surgeon- 
General  Walter  Wyman,  and  represented  the  dried  standard  toxin 
which  is  used  in  the  standardization  of  tetanus  antitoxin  in  the 
Hygienic  Laboratory  and  furnished  to  the  manufacturers  of  anti- 
toxin. In  the  determination  of  the  value  of  the  antitoxin  the  L  + 
dose  is  the  test  dose  of  the  toxin.  The  L+  dose  is  the  smallest 
quantity  of  tetanus  toxin  that  will  neutralize  one-tenth  of  an  immu- 
nity unit,  plus  a  quantity  of  toxin  sufficient  to  kill  an  animal  in  just 
four  days.  The  L+  dose  of  the  toxin  which  was  used  in  these  tests 
contained  100  minimal  lethal  doses  (MLD)  for  a  350-gram  guinea  pig. 
The  toxin  was  kept  in  a  dark,  cold  place  and  during  the  course  of 
these  tests  its  virulence  was  controlled  by  repeated  tests  with  an 
antitoxin  of  known  value. 

The  guinea  pigs  used  in  connection  with  this  standardization  were 
carefully  selected,  being  vigorously  healthy  animals  of  from  350  to 
370  grams  in  weight.  The  glassware  employed  in  the  work  was 


16 


VETERINARY    TETANUS   ANTITOXIN. 


selected  according  to  the  recommendation  of  Rosenau,  and  was  not 
used  for  any  other  purpose.  The  mixing  cylinders  and  mixing  flasks 
were  the  kind  designed  by  Rosenau.  Ehrlich's  delivery  pipettes 
graduated  into  hundredths,  as  well  as  special  delivery  and  capacity 
pipettes,  were  used.  The  syringes  employed  were  a  modification  of 
the  old  Koch  syringe,  in  which  the  barrel  tapers  gradually  to  the 
needle,  so  that  the  last  drop  runs  out  readily. 

The  necessary  measures  against  bacterial  contamination  were  taken 
during  the  execution  of  the  work.  All  the  glassware  was  first  ren- 
dered chemically  clean  and  then  sterilized  for  one  hour  at  120°  C. 
All  the  various  steps  in  the  procedure  were  followed  carefully  in  order 
that  the  results  would  be  accurate  and  reliable.  The  method  of  the 
standardization  was  executed  in  every  particular  as  described  by 
Rosenau  and  Anderson  in  Bulletin  43  of  the  Hygienic  Laboratory. 

The  number  of  immunity  units  contained  in  a  cubic  centimeter  of 
serum  is  determined  by  the  quantity  of  antitoxin  which  saved  the 
life  of  a  guinea  pig  for  ninety-six  hours  against  the  official  test  dose 
of  the  toxin.  This  quantity  of  antitoxin  represents  one-tenth  of  an 
immunity  unit.  For  instance,  if  a  guinea  pig  receiving  0.0015  c.  c. 
of  the  antitoxin  with  the  official  test  dose  of  toxin  is  saved  for  ninety- 
six  hours,  this  quantity  of  antitoxin  contains  one-tenth  of  an  immu- 
nity unit.  Accordingly,  the  following  equation  is  represented: 
0.0015  :0.1  :  :  1  :  x,  which  indicates  that  1  cubic  centimeter  of  the 
serum  contains  66  units. 

In  the  following  tables  are  given  the  results  of  the  standardization 
obtained  from  the  tests  of  the  various  samples  of  veterinary  tetanus 
antitoxin  examined  in  this  laboratory: 

TABLE  1. — H.  K.  Mulford  Company's  Tetanus  Antitoxin  Serum  for  rtttrinary  use. 
Immunizing  dose,  labeled  to  contain  500.000  units.  To  be  exchanged  after  Mat/  1, 1910. 
Laboratory  -Yo.  i960.  Syringe  contained  7.6  c.  c. 


Xo.  of 
guinea 
pig- 

Subcutaneous  in- 
jection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Time  of  death  or  effect  of  injection. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1 

e.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 
.0006 
.0006 
.0006 
... 

c.  c, 

a  ooio 

.0011 
.0012 
.0013 
.0014 
.0015 
.0020 
.0025 
• 

1  dav.  30  hours. 
1  day.  26  hours. 
3  days,  19  hours. 
5  <1avs  22  hours  .  , 

81 

615 

2  

3 

4 

5 

Symptoms. 
X"o  symptoms. 

DO. 

Da 
Do. 

6  

8... 

9... 

RESULTS  OF   TESTS. 


17 


TABLE  2. — Parke,  Davis  &  Co.'*  Antitetanic  Serum  (veterinary).     To  be  exchanged  after 
November  24,  1910.     Unit  value  not  stated.     Syringe  contained  JO  c.  c. 


No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1  

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0006 
.0007 
.0008 
.0009 
.0010 
.0015 
.0020 
.0025 
•    .0030 

1  day,  12  hours. 
3  days,  7  hours. 
4  days,  12  hours.... 

125 

1,250 

2  

3  

4        .   . 

Symptoms. 
Slight  symptoms. 
No  symptoms. 
Do. 
Do. 
Do. 

5  

6  

7  

8 

9  

TABLE  3. — H.  K.  Mulford  Company's  Tetanus  Antitoxin  Serum  for  veterinary  use. 
Immunizing  dose,  labeled  to  contain  500,000  units.  To  be  exchanged  May  15,  1910. 
Laboratory  No.  2960.  Syringe  contained  7.5  c.  c. 


Subcutaneous  in- 
jection. 

Antitoxin  units. 

guinea 

Time  of  death  or  effect  of  injection. 

Number 

Total 

Pig- 

Toxin. 

Anti- 
toxin. 

per  cubic 
centi- 

number 
in 

meter. 

syringe. 

c.  c. 

c.  c. 

-• 

1... 

0.0006 

0.0010 

2  days,  12  hours. 

2 

.0006 

.0013 

4  days,  7  hours  ..           .     .                     

81 

607 

3  .... 

.0006 

.0016 

6  days,  8  hours. 

4  

.0006 

.0019 

Slight  symptoms. 

6  

.0006 

.0022 

No  symptoms. 

TABLE  4.  —  Pasteur  Laboratories,  Paris,  France,  Antitetanic  Serum  for  veterinary  use. 
Unit  value  not  stated.     Bottle  contained-  10  c.  c. 

No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
bottle. 

1  

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0010 
.0013 
.0016 
.0019 
.0022 

4  days,  2  hours  

100 

1,000 

2  

Slight  symptoms. 
No  symptoms. 
Do. 
Do. 

3  

4  

5  

18 


VETERINARY   TETANUS  ANTITOXIN. 


TABLE  5. — Lederle  Antitoxin  Laboratories,  Tetanus  Antitoxin  (veterinary').  Immuniz- 
ing dose,  labeled  to  contain  1,500  units.  Laboratory  No.  19 A.  To  be  exchanged  June 
21,  1910.  Syringe  contained  10.4  c.  c. 


No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1... 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.00035 
.00050 
.00065 
.00080 
.00085 

1  day,  18  hours. 
1  day,  15  hours. 
4  days,  3  hours 

154 

1,601 

2  

3 

4  

Symptoms. 
No  symptoms. 

5 

TABLE  6.  —  Parke,  Davis  &  Go's  Antitetanic  Serum  (veterinary).     To  be  exchanged  after 
December  8,  1910.     Unit  value  not  stated.    Syringe  contained  10  c.  c. 

No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1... 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0005 
.0008 
.0011 
.0014 
.0017 

3  days,  1  hour. 
5  days,  20  hours 

125 

1,250 

2 

3 

No  symptoms. 
Do. 
Do. 

4  

5 

TABLE  7. — Parke,  Davis  &  Go's  Antitetanic  Serum  (veterinary).     To  be  exchanged  after 
December  8,  1910.     Unit  value  not  stated.    Syringe  contained  10  c.  c. 


Subcutaneous  in- 
jection. 

Antitoxin  units. 

guinea 

Time  of  death  or  effect  of  injection. 

Number 

Total 

Pig- 

Toxin. 

Anti- 
toxin. 

per  cubic 
centi- 

number 
in 

meter. 

syringe. 

c.  c. 

c.  c. 

1 

0.0006 

0.0005 

2  days,  21  hours. 

2 

.6006 

.0008 

Symptoms 

125 

1  250 

3 

.0006 

.0011 

Slight  symptoms. 

4  

.0006 

.0014 

No  symptoms. 

5 

.0006 

.0017 

RESULTS  OP  TESTS. 


19 


TABLE  8. — Parke,  Davis  &  Co's  Antitetanic  Serum  (veterinary).     To  be  exchanged  after 
January  14,  1911.     Unit  value  not  stated.     Syringe  contained  10  c.  c. 


No.  of 
guinea 
pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1... 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0010 
.0014 
.0018 
.0022 
.0026 

4  days,  5  hours  

100 

1,000 

2 

Slight  symptoms. 
No  symptoms. 
Do. 
Do. 

3  

4  

5    .     . 

TABLE  9. — H.  K.  Mulford  Company's  Tetanus  Antitoxin  Serum  for  veterinary  use.  Im- 
munizing dose,  labeled  to  contain  500,000  units.  To  be  exchanged  after  May  15,  1910. 
Laboratory  No.  2971.  Syringe  contained  8  c.  c. 


No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0008 
.0011 
.0014 
.0017 
.0020 

2  days,  1  hour. 
2  days,  18  hours. 
2  days,  23  hours. 
6  days,  4  hours                                               .        

59 

472 

2  

3 

4 

5  

Slight  symptoms. 

TABLE  10. — Pasteur  Laboratories,  Paris,  France,  Antitetanic  Serum  for  veterinary  use. 
Unit  value  not  stated.     Bottle  contained  10  c.  c. 


No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
bottle. 

1... 

c.  c. 
0,0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0006 
.0009 
.0012 
.0015 
.0018 

2  days,  20  hours. 
6  days,  7  hours 

Ill 

1,110 

n 

g 

Symptoms. 
No  symptoms. 
Do. 

4... 

5     . 

VETERINARY   TETANUS   ANTITOXIN. 


TABLE  11. — Pasteur  Laboratories,  Paris,  France,  Antitetanic  Serum  for  veterinary  use. 
Unit  value  not  stated.     Bottle  contained  10  c.  c. 


No.  of 
guinea 
Pig. 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
bottle. 

1 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.00050 
.00065 
.00080 
.00095 
.00110 

3  days,  6  hours. 
4  days,  18  hours  

153 

1,530 

2 

3 

No  symptoms. 
Do. 
Do. 

4  

5 

TABLE  12. — H.  K.  Mulford  Company's  Tetanus  Antitoxin  Serum  for  veterinary  use. 
Immunizing  dose,  labeled  to  contain  500,000  units.  To  be  exchanged  after  May  1, 1910. 
Laboratory  No.  2,960.  Syringe  contained  7.5  c.  c. 


No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1... 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.0007 
.0010 
•    .0013 
.0016 
.0019 

1  day  14  hours. 
3  days  7  hours. 
Symptoms 

81 

607 

2 

3 

4 

No  symptoms. 
Do. 

5 

TABLE  13.  —  Lederle  Antitoxin  Laboratories,  Tetanus  Antitoxin  (veterinary).     Immuniz- 
ing dose,  labeled  to  contain  1,500  units.     Laboratory  No.  19  A.     To  be  exchanged  June 
21,  1910.     Syringe  contained  8.5  c.  c. 

No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection. 

Time  of  death  or  effect  of  injection. 

Antitoxin  units. 

Toxin. 

Anti- 
toxin. 

Number 
per  cubic 
centi- 
meter. 

Total 
number 
in 
syringe. 

1... 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.00024 
.  0003333 
.00043 
.00053 
.00060 

1  day  16  hours. 
2  days  22  hours. 
Symptoms          .                             

232 

1,972 

2 

3 

4 

No  symptoms. 
Do. 

5  

DISCUSSION    OF    RESULTS. 


21 


TABLE  14. — Pasteur  Laboratories,  Paris,  France,  Antitetanic  Serum  for  veterinary  use. 
Unit  value  not  stated.     Bottle  contained  10  c.  c. 


No.  of 
guinea 
Pig- 

Subcutaneous  in- 
jection.         . 

Time  of  death  or  effect  of  injection. 

Antitox 

Number 
per  cubic 
centi- 
meter. 

n  units. 

Total 
number 
in 
bottle. 

Toxin. 

Anti- 
toxin. 

1... 

c.  c. 
0.0006 
.0006 
.0006 
.0006 
.0006 

c.  c. 
0.00035 
.00050 
.00065 
.00080 
.00095 

1  day  14  hours. 
2  days  20  hours. 
4  days  5  hours  

153 

1,530 

2  

3 

4.   .. 

4  days  16  hours. 
Slight  symptoms. 

5  

The  results  of  these  tests  clearly  demonstrate  the  variations  in  the 
potency  of  veterinary  tetanus  antitoxins  at  present  on  the  market. 
While  the  preparations  of  the  individual  manufacturers  do  not  show 
such  marked  differences  in  strength,  yet  they  do  not  uniformly  con- 
tain a  sufficient  number  of  units. 

In  accordance  with  the  law  of  1902,  the  manufacturers  of  human 
antitoxins  are  required  to  state  on  the  labels  of  the  packages  the 
number  of  units  that  the  particular  antitoxin  possesses.  Should  it 
be  found  on  investigation  that  the  antitoxin  does  not  come  to  within 
10  per  cent  of  the  strength  stated  on  the  labels,  the  manufacturer  is 
immediately  required  to  recall  from  the  market  all  that  particular 
antitoxin.  Thus,  if  a  physician  intends  to  use  the  antitoxin  either 
for  immunizing  purposes  or  as  a  curative  agent,  he  is  accurately 
guided  in  the  dose  by  the  statement  on  the  label.  It  does  not  mat- 
ter who  the  manufacturer  of  the  antitoxin  is.  This  latter  fact  is 
in  itself  also  of  great  importance,  as  frequently  the  drug  stores  carry 
antitoxins  of  only  one  or  two  manufacturers. 

On  the  other  hand,  the  veterinarian  has  not  always  the  good  for- 
tune of  knowing  the  number  of  units  that  an  antitoxin  which  he 
purchases  contains.  Only  one  pf  the  manufacturers  states  on  the 
label  the  number  of  American  units  contained  in  his  veterinary  anti- 
toxin. One  other  manufacturer  still  uses  for  his  veterinary  antitoxin 
a  standardization — other  than  American— by  which  he  can  label  his 
product  in  the  hundred  thousands,  yet  his  antitoxin  for  the  human 
gives  the  units  in  the  American  standard.  Why  should  two  different 
standards  be  maintained,  one  for  the  human  and  the  other  for  veter- 
inary antitoxin? 

The  veterinary  tetanus  antitoxins  are  marketed  in  immunizing 
doses  and  curative  doses.  The  immunizing  dose  is  supposed  to  con- 
tain 1,500  American  units.  The  volumetric  quantity  of  this  dose 
was  found  to  be  accurately  10  c.  c.  in  the  Parke,  Davis  &  Co.  and  the 
Pasteur  product,  while  the  Lederle  serum  contained  from  8.5  c.  c. 
to  10.4  c.  c.  and  the  Mulford  syringe  from  7.5  to  8  c.  c.  Now,  should 


22  VETERINARY   TETANUS  ANTITOXIN. 

it  be  desired  to  administer  to  a  horse  an  immunizing  dose  of  the 
antitoxin  it  can  readily  be  seen  from  the  results  obtained  in  these 
tests,  as  indicated  by  the  tables,  that  while  the  immunizing  dose  of 
some  of  the  serums  contains  the  desired  1,500  units,  others,  on  the 
other  hand,  possess  less  than  one-third  of  that  strength.  For  instance, 
according  to  the  test  shown  by  Table  9,  the  syringe  contained  8  c.  c. 
of  serum  with  59  units  per  cubic  centimeter;  thus  the  immunizing 
dose  in  this  case  represents  only  472  units,  and  of  course  the  curative 
dose  is  correspondingly  low.  This  alone  is  sufficient  to  indicate  the 
urgent  necessity  for  some  uniformity  in  standardizing  the  veterinary 
antitoxins,  and  also  for  Federal  legislation  by  which  they  could  be 
subjected  to  a  periodical  control  with  reference  to  their  potency. 

Under  the  present  conditions  there  is  the  constant  uncertainty 
regarding  the  strength  of  the  serum,  as  the  veterinarian  has  no  assur- 
ance whatever  of  its  potency,  and  is  solely  dependent  on  the  relia- 
bility of  the  manufacturer. 

CONCLUSIONS. 

1.  The  veterinary  tetanus  antitoxins  prepared  by  the  different 
manufacturers  have  not  a  uniform  potency,  and  the  variation  amounts 
in  some  instances  to  about  two-thirds  less  than  the  strength  which 
it  should  possess. 

2.  In  order  to  insure  a  uniform  strength,  the  manufacturers  of 
veterinary  tetanus  antitoxins  should  be  required  to  use  the  American 
standard,  and  to  state  on  the  label  the  number  of  American  units 
the  dose  contains,  as  is  required  for  human  tetanus  antitoxin. 

3.  The  immunizing  dose  for  a  horse  should  contain  at  least  1,500 
immunity  units  of  the  standard  established  by  the  United  States 
Public  Health  and  Marine-Hospital  Service. 

4.  It  is    seen  that  the  veterinary  tetanus    antitoxins  vary  ex- 
travagantly in  the  unit  strength,  and  some  are  comparatively  weak 
in  antitoxic  potency,  which  shows  the  necessity  for  the  same  super- 
vision by  the  United  States  Department  of  Agriculture  over  biologi- 
cal products  used  in  veterinary  medicine  as  is  now  exercised  by  the 
United  States  Public  Health  and  Marine-Hospital  Service  over  simi- 
lar products  used  in  human  medicine. 

5.  The  request  for  such  supervision  should  have  the  indorsement 
of  the  veterinarians  and  live-stock  interests  of  this  country. 

o 


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